• Cryopreservation of zebrafish (Danio rerio) blastomeres using controlled slow cooling

      Creamer, Delta Patricia Menendez (University of Bedfordshire, 2008-07)
      Cryopreservation of aquatic species has been widely studied especially zebrafish gametes, embryos and larvae. Cryopreservation of blastomeres has the advantage of preserving both paternal and maternal genetic information. The research work presented in this thesis investigated the toxicity of cryoprotectants to 75% epiboly stage blastomeres of zebrafish (Danio rerio) before cryopreservation. DMSO was found to be the least toxic cryoprotectant for blastomeres, after 30 min incubated in PBS at room temperature, with 97.8% survivaL Cryopreservation of zebrafish blastomeres was carried out using controlled slow cooling. In addition to cryoprotectant, the cryoprotective properties of other compounds were also investigated including NaHC03, coffee and honey, and their solutions were used as cryopreservation media. Comparison of blastomere survival in different media after freezing showed honey to be the most effective, with 98.1 % survival immediately after freeze thawing. Comparison of blastomers cryopreserved in honey and DMSO, after 60 min incubation in PBS at room temperature following freeze-thawing and cryoprotectant removal, showed honey to be the more effective cryoprotectant for controlled slow cooling of zebrafish blastomeres with 97.2% survival. Results from the 'present study showed honey to have properties that protect the blastomere from freezing injury.
    • Development of new methods to assess the quality of zebrafish (Danio rerio) ovarian follicles

      Zampolla, Tiziana (University of BedfordshireUniversity of Bedfordshire, 2009-03)
      High quality fish oocytes are essential for in vitro maturation (IVM), in vitro fertilization (IVF) protocols, and for use in cryopreservation. It is important to develop methods for assessing oocyte quality for applications in aquaculture, the preservation of endangered species and managing fish models used in biomedical research. The lack of reliable methods of evaluating oocyte quality limits progress in these areas. The present study was undertaken to develop new methods to assess ovarian follicle viability and quality of stage III zebrafish (Danio rerio) ovarian follicles. The methods developed were then applied to study the impact of cryoprotectant and/or cryopreservation procedures. A vital staining procedure, not previously used with zebrafish oocytes, has been investigated. FDA-PI (Fluorescein diacetate-Propidium Iodide) staining was found to be a more sensitive then currently used viability tests and it could also be applied to all ovarian follicles developmental stages. Mitochondrial activity and distribution as biological markers was investigated with the mitochondrial membrane potentialsensitive dye JC-1- (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide). Confocal microscopy, Cryo-scanning and electron microscopy studies were undertaken to determine mitochondria distributional arrangement within the ovarian follicle. This provided new information on zebrafish ovarian follicle structure, and showed that mitochondria exhibited a contiguous distribution at the margin of the granulosa cell layer surrounding stage III zebrafish oocytes. Cryoscanning results showed a polygonal structure of the vitelline envelope, which is reported here for the first time with the mitochondrial distributional arrangement in the granulosa cell layer. Mitochondrial distribution and the evaluation of mitochondrial activity proved to be sensitive markers for ovarian follicle quality, providing more detailed information on cryoprotectant impact. The measurement of ATP levels, ADP/ATP ratio and mtDNA copy number were also undertaken following cryoprotectant exposure. These findings, together with the observation of mitochondrial distribution, suggested that even cryoprotectant treatments that are considered to have little or no toxicity can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development. Therefore, a further optimization of the currently used protocol may need to be considered. The study of organelle distribution and organisation would support in vitro maturation and oocyte development fields, as well as their use as biological markers for quality determination. These findings will contribute to a better understanding of oogenesis/folliculogenesis processes in fish.
    • Isolation and culturing of zebrafish pluripotent cells

      Sana, Salma (University of Bedfordshire, 2012-10)
      Zebrafish (Dania rerio) is an important model organIsm for the studies of vertebrate development and gene expression in the field of molecular biology and biomedicine. Its embryonic stem cells provide a unique tool linking in vitra and in vivo genetic manipulations of animal genomes. The aim of the project was to determine the most suitable embryo stage for the isolation and culturing of pluripotent cells of zebrafish embryos. Studies were carried out to investigate the expression of two pluripotency markers i.e. Oct4 and Sa1l4 at certain embryonic stages employing Immunohistochemistry. The protein expression studies indicated maximum expression of Oct4 and Sa1l4 at high stage. Primary cultures were initiated from zebrafish high stage embryos in basal nutrient medium; supplemented with insulin, selenite, epidermal growth factor and Foetal Bovine serum. Experiments were conducted for the determination of optimum concentrations of FBS. The growing cultures identified the signs of differentiation i.e. melanocytes and neurite formation. Basic fibroblast growth factor (bFGF) was found to inhibit the differentiation in cultures. The developed pluripotency markers were tested on cultured embryonic cells. Results indicated that these markers work well for the identification ofphenotype of embryonic cell colonies in zebrafish. The establishment of pluripotent cell line will enable knock out and knock in technologies to be used in this species and have important applications in functional genomics research.
    • Storage and subsequent cryopreservation of liver cells of zebrafish

      Phonphaisan, Wirada (University of Bedfordshire, 2008)
      When gametes or embryos are not available or cannot be successfully cryopreserved, somatic cells should be considered for cryobanking of material from endangered fish species. Although the preservation of such material is not difficult, their collection and ultimate cryopreservation can present logistic difficulties; and initial storage is probably needed to retain viability before cryopreservation. The aim of this study was to develop a reliable method for initial storage and cryoprotectant treatment of liver cells that could be employed prior to the cryopreservation. Two bathing solutions were assessed for initial storage at 4°C or 6°C over a 48 hour storage period in chilling sensitivity studies. Medium C (lml L~15 supplemented with Hepes (5.958g1L), NaHC03 (0.42g/L), Gentamycine (lOOmglL), Amphotericine B (2.5mgIL), Lglutamine (0.374g/L) and Fetal bovine serum (10%)) and PBS were used as bathing solutions of the cells stored at 4°C or 6°C for up to 48 hours. Fluorescein diacetate propidium iodide staining was used to assess the viability after 12,24 and 48 hours of storage. Four cryoprotectants (CPAs) were investigated at different concentrations-DMSO (lM), MeOH (2M, 3M and 4M), PG (lM) and sucrose (0.05M and O.IM)and used to treat the cells for up to 30 minutes. Viability assessment was done using Trypan blue and Fluorescein diacetate - propidium iodide staining after 10, 20 or 30 minute exposure to CP As. Fresh and stored liver cells were frozen using slow cooling methods based on existing LIRANS procedures for cryopreservation of zebrafish blastomeres. The results indicated that exposure to 4·C PBS for 12, hours was an optimal condition for storage of liver cell prior to cryopreservation. In addition, results showed that cryopreservation at a slow freezing rate following 10 minute treatment of 2M methanol, allowed the recovery of more than 50% of the zebrafish liver cells.
    • Studies on cryopreservation of early stage zebrafish (Danio rerio) oocytes using controlled slow cooling

      Nwankwo, Judith N. (University of Bedfordshire, 2007-11)
      In the last three decades, much effort has been made toward the successful cryopreservation of fish gaments. The spermatozoa from many species including salmonid, cyprinids, and acipenseridae has been successfully cryopreserved and well documented. Systematic studies on fish oocytes cryopreservation has not been carried out until recently. In this study, the effect of cryoprotectant toxicity on stage I and II zebrafish oocytes were studied and the controlled slow cooling was used at different cooling rates to identify the optimal cooling rate for these oocytes and the optimal conditions for removing cryoprotectant. Methanol was found to be the least toxic CPA for zebrafish oocytes and 4M methanol in potassium chloride (KCI) buffer was used as the cryoprotective solution. Oocytes were cooled with programmable cooler (Planner KRYO 550) using controlled slow cooling at 0.3, 0.5 and 1°C/min rates, combined with seeding at -12.5°C and plunge into liquid nitrogen at -80°C. 1°C/min was identified to be the optimal condition for cryopreservation of stage I and II oocytes. High survival rate were obtained after cooling to -196°C assessed with TB staining which were 87.8 ± 4.9% and 86.8 ± 6.5% for stage I and II respectively. It was also found in this study that recovery conditions affect oocyte survivals after freezing. Oocytes which had been incubated for 60 or 120min in room temperature showed higher survival rate than those assessed immediately after freezing. This study has shown that stage II (cortical alveoli stage) oocytes were more resistible to freezing than stage I (primary growth stage) oocytes. More oocytes viability studies are needed to validate these results.